The present invention relates generally to the disease of anthrax and, more particularly, to a novel vaccine strain of Bacillus anthracis. Anthrax infections are initiated by spores of Bacillus anthracis, a gram-positive, rod-shaped bacterium found in soil. Bacillus anthracis spores do not divide, have no measurable metabolism and are markedly resistant to biological extremes of heat, cold, pH, desiccation, chemicals and irradiation. In the spore form, Bacillus anthracis survives for decades, perhaps centuries. Domestic livestock are frequent victims of the disease, but human cases of anthrax can occur as a result of exposure to infected animals or animal products. Anthrax has also been recognized as a likely biological warfare or terrorist agent.
Anthrax is a complex, poorly understood disease. The pathogenic process of anthrax and the mechanisms of immunity to the disease have not been completely defined. All known anthrax virulence genes are expressed by the vegetative form of Bacillus anthracis that results from the germination of spores within the body of the host. Spores introduced into the body by abrasion, inhalation or ingestion are phagocytosed by macrophages and carried to regional lymph nodes. Spores germinate inside the macrophage and become vegetative bacteria. After germination and local multiplication within the macrophage, the vegetative bacteria kill the macrophage and are released into the bloodstream, reaching high numbers (up to 108 organisms per milliliter of blood) and causing massive septicemia. It is believed that no immune response is initiated against vegetative bacilli once they have been released from the macrophage.
It is believed that anthrax bacilli express a range of virulence factors. The two major factors are a tripartite toxin and an antiphagocytic capsule composed of poly-D-glutamic acid. Anthrax toxin and capsule genes are apparently expressed early after germination within the macrophage. The resulting toxemia and bacteremia have systemic effects that lead to the death of the host.
The major virulence factors of Bacillus anthracis are encoded on two virulence plasmids, pX01 and pX02. The toxin-bearing plasmid, pX01, is 184.5 kilobase pairs (kbp) in size and codes for the genes (cya, lef, and pagA) that make up the secreted toxins. Regulation of toxin production is also encoded on pX01; it contains transacting regulator genes atxA and atxR.
The three proteins of the toxin are protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase. EF is a calmodulin-dependent adenylate cyclase which causes fluid loss through elevation of cellular cAMP concentrations in affected tissues. Neither LF or EF are toxic alone; they can produce deleterious effects only when combined with PA, so named because of its use in the protective anthrax vaccine. Following the A-B model of toxicity, PA serves as a necessary carrier model for LF and EF and permits penetration into host cells. Lethal toxin, which results from the combination of LF+PA, stimulates the macrophages to release the shock-inducing mediators, necrosis factor α and interleukin-1β, which are partly responsible for sudden death in systemic anthrax. Edema toxin, which results from the combination of EF+PA, is responsible for the massive edema seen in anthrax. Edema toxin also plays a role in inhibiting phagocytic and oxidative burst activities of polymorphonuclear leukocytes. Bacterial toxins that increase cAMP tend to decrease the immune response of phagocytes, thereby contributing to the development of infection.
The smaller capsule-bearing plasmid, pX02, is 95.3 kbp in size and codes for the genes (capB, capC, capA) involved in the synthesis of the polyglutamyl capsule. pX02 also encodes for a known transacting regulating gene for capsule modulation, acpA. atxA also appears to regulate acpA transcription to some degree.
The capsule is weakly antigenic and antiphagocytic. The toxins are thought to inhibit the immune response mounted against infection while the capsule inhibits phagocytosis of vegetative anthrax bacilli.
In addition to the major virulence factors already described, Bacillus anthracis likely expresses other plasmid- and chromosome-encoded genes that contribute to the pathogenisis of the organism. Identification of other genes contributing to virulence is crucial to the further development of effective protection against anthrax.
Expression of the known major virulence factors previously discussed (tripartite toxin and capsule) appears to be regulated by two host-specific cues: elevated temperature and carbon dioxide/bicarbonate concentration. During in vitro growth of Bacillus anthracis, synthesis of toxin protein and capsule is greatest when cultures are incubated at elevated (5% or greater) atmospheric CO2 or when bicarbonate is added to culture medium in a closed vessel. Toxin and capsule synthesis is also increased when cultures are incubated at 37° C. compared to when they are incubated at 28° C. CO2/bicarbonate and temperature-controlled gene expression is at the level of transcription. As indicated previously, regulation of the expression of the toxin and capsule genes is mediated by the transcriptional activator atxA; expression of the capsule gene is also controlled by transcriptional regulator acpA.
The effect of these signals (CO2/bicarbonate concentration and temperature) in culture medium may be compared with their physiological role in mammalian hosts; concentrations of CO2 and bicarbonate in humans are similar to those that activate toxin and capsule production in vitro, and the same is true of human body temperature. It is believed that these signals play similar roles in vitro and in vivo by providing an optimal environment for expression of known Bacillus anthracis toxin and capsule genes.
As indicated previously, the loss of either plasmid pX01 or pX02 results in a marked reduction of virulence. This forms the basis for effective vaccine production. Historically, vaccine strains of anthrax bacteria were made by rendering virulent strains free of one or both plasmids. Pasteur, a heat-attenuated, pX02-carrying strain is encapsulated but does not express toxin components (pX01−/pX02+). Sterne, an attenuated strain that carries pX01, can synthesize toxin but does not have a capsule (pX01±/pX02−).
It is frequently convenient to class Bacillus anthracis with the “Bacillus cereus group” of bacilli which on the basis of phenotype comprises Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, and Bacillus mycoides. Except for Bacillus anthracis, all members of this group are resistant to penicillin. Bacillus anthracis is easy to differentiate from other member of the Bacillus cereus group by observing the morphological features of the colony on nutrient or blood agar plates. Colonies of most Bacillus anthracis isolates have a matt appearance, are fairly flat, markedly tacky, white or grey-white and non-hemolytic on blood agar and often having curly tailing at the edges. The unusually tenacious colonies are able to retain their shape when manipulated; disturbed sections of the colony often stand up like “beaten egg whites.” Bacillus anthracis is non-motile, sensitive to penicillin and the diagnostic Cherry gamma phage and able to produce the capsule in blood or on nutrient agar containing 0.7% bicarbonate following incubation in a 5-20% CO2 atmosphere.
In practical terms, the demonstration of virulence constitutes the principle point of difference between typical strains of Bacillus anthracis and those of other members of the Bacillus cereus group. However, there is evidence that the virulence plasmids can be transferred between the Bacillus cereus group species through genetic engineering, although it is not clear how stable the resulting hybrids are.
An anthrax vaccine for humans is approved for use in the United States by the Food and Drug Administration. Designated anthrax vaccine adsorbed (AVA), it is an aluminum-hydroxide-precipitated preparation of PA from attenuated, nonencapsulated Bacillus anthracis cultures of the Sterne strain. The anthrax vaccination protocol consists of 3 subcutaneous injections given 2 weeks apart followed by 3 additional subcutaneous injections given at 6, 12 and 18 months. Annual booster injections of the vaccine are required to maintain immunity. Mild local reactions consisting of slight tenderness and redness at the injection site can occur in approximately 30% of recipients. Severe local reactions occur infrequently and consist of extensive swelling of the forearm in addition to the local reaction. Systemic reactions characterized by flu-like symptoms occur in fewer than 0.2% of vaccines.
Animal studies have shown that AVA affords protection against inhalational anthrax and a limited trial of a similar vaccine in humans indicated that it afforded considerable protection against cutaneous anthrax. Studies have also demonstrated, however, that the live Sterne spore veterinary vaccine is more protective than the human chemical vaccine. The enhanced protection conferred by the live vaccine probably results from stimulation of the host cellular immune system concurrent with the humoral response to PA. The main limitation of the Sterne vaccine is safety. Its use is sometimes associated with tissue necrosis at the site of inoculation and there have been rare fatalities. Because of these safety concerns, spore vaccines have generally not been used for humans.
The established virulence factors of Bacillus anthracis have been the targets of most attempts to develop vaccines. As indicated previously, PA is asserted to be the essential anthrax-derived antigen for the protective action of the current vaccine. Nevertheless, studies have repeatedly demonstrated that titers to PA do not correlate strictly with the level of immunity to anthrax. Moreover, it is important to note that antibodies to PA induced by the vaccine are directed against the action of the toxin and not at the multiplying Bacillus anthracis in an infection. It has also been postulated that Bacillus anthracis strains could be created by adding foreign genes from other toxic organisms. As indicated previously, studies have shown that virulence plasmids can be transferred between the Bacillus cereus group of organisms.
Clearly there is a need for new candidate antigens for vaccine development, especially those that act prior to expression of anthrax toxins into the body. Such vaccines should also be effective against infection with strains that have been engineered with additional toxins.
Critical to development of effective protection against anthrax is an understanding of the initial pathogenesis of the disease and its virulence mechanisms. Events occurring during the initial moments when bacterial pathogens first encounter the host are critical for successful establishment of infectious loci. The pathogenesis of anthrax appears to be related primarily to the unique sensitivity of the macrophage to the activity of lethal toxin, in addition to the adenylate cyclase activity of edema toxin and the antiphagocytic properties of the capsule. As indicated previously, the genes for these virulence factors are induced in response to specific host-related cues, that is, CO2/bicarbonate levels and physiological body temperature. There is a need for a vaccine directed to Bacillus anthracis targets vital for early steps in the infection process, containing antigens which elicit antibodies targeted at the spore or germinating cell.
It is therefore a principal object of the present invention to provide a vaccine strain of Bacillus anthracis from which may be produced an improved anthrax vaccine which is safe, nonreactogenic, efficacious against genetically engineered strains, and which requires a minimal number of injections to achieve and maintain long-term immunity. It is a further object of the invention to provide a vaccine strain of Bacillus anthracis that will enable identification of new genes that contribute to the pathogenesis of the organism and thereby elucidate new antigens that play a role in eliciting a specific, protective immune response early in the infection process.